During spermatogenesis, regional specialization of the plasma membrane occurs resulting in a mature gamete with distinctly polarized functional and biochemical properties. One feature of this differentiative process is the appearance of novel surface constituents in a precise temporal sequence. Both immunological and biochemical techniques have been used to characterize germ cell-specific constituents and monitor membrane assembly. Central to these studies are methods for the purification of germ cells at defined stages of spermatogenesis by enzymatic dissociation of adult or prepuberal testes followed by unit gravity sedimentation. Three areas of research have been pursued: (a) Polyclonal and monoclonal antibodies have been used to characterize macromolecules first appearing on the surface of pachytene spermatocytes, coincident with a period of maximal protein synthesis. These constituents are not shared by most somatic cells and include at least ten proteins, a probable lipid constituent, and large polylactosamine glycoconjugates. Some of these components are retained on sperm, restricted to distinct domains on the cell surface. (b) Conditions for the short term culture of spermatogenic cells are being tested. Earlier meiotic stages from 17-day-old animals exhibit more stringent requirements for in vitro maintenance than later stages from adult mice. Media supplementation with growth factors and hormones has resulted in increased viability of the prepuberal germ cells and should facilitate metabolic studies and the development of in vitro functional assays. (c) Protein synthesis in isolated spermatogenic cells has been examined by 2D PAGE and autoradiography following short term culture with [35S]methionine. Synthetic profiles become more complex throughout meiosis. A number of proteins previously identified as surface antigens are synthesized in a stage-specific manner. Germ cell surface constituents exhibiting both tissue and stage specificity are candidates for further studies exploring cell-cell interactions during spermatogenesis and fertilization.